Effects of different freezing techniques on the rejection of allogeneic vascular transplantation / 中国组织工程研究

BACKGROUND:Cryopreservation can better ensure the integrity of the vascular structure.How to reduce its immunogenicity to improve rejection after transplantation has attracted more and more attention. OBJECTIVE:To review the research progress of freezing treatment to reduce vascular immunogenicity after allogeneic vascular transplantation in order to provide a reference for clinical research. METHODS:A systematic search of Chinese and English databases CNKI,WanFang and PubMed,as well as online websites Baidu and Google Scholar since the establishment of the database has published literature on reducing vascular immunogenicity after allogeneic vascular transplantation.Keywords were"cardiovascular disease,endothelial cells,cryopreservation,blood vessel transplantation or vascular graft,immunogenicity,immune rejection,allograft or allogeneic transplantation or allograft transplantation and cryoprotectant".A total of 68 articles were included according to the inclusion and exclusion criteria. RESULTS AND CONCLUSION:(1)The review found that the rejection of allogeneic vascular transplantation can be reduced by improving the existing freezing technology,which mainly involves the selection of freezing and thawing methods and cryoprotectants.(2)The existing research suggests that the freeze-drying method is superior to the low-temperature cryopreservation,but due to the limited conditions,it is still dominated by low-temperature cryopreservation.Among them,vitrification cryopreservation,slow rewarming and the use of stainless steel and even silver-containing materials are better than programmed cryopreservation and rapid rewarming.(3)The combined selection of permeable and non-permeable cryoprotectants can further reduce the occurrence of rejection while reducing their toxicity.

Vitrification Preservation of Oocyte Hydrogel Microspheres Prepared by Microfluidics / 生物化学与生物物理进展

ObjectiveThis study aimed to develop a novel method for encapsulating oocytes in sodium alginate hydrogel using microfluidics, then to vitrify these encapsulated oocytes in a single-step process with low concentrations of cryoprotectants. MethodsWe utilized a flow-focusing microfluidic chip to generate sodium alginate hydrogel microspheres. The influence of various parameters, including throat structure, cross-linking method, sodium alginate concentrations, and flow rate ratios on the stability diameter, and coefficient of variation of microspheres were examined. To further investigate the cold-resistance of these microspheres, we used cryomicroscopy to observe changes in volume and morphology of microspheres during cooling and warming processes. We used microfluidic chip to encapsulate oocytes in sodium alginate hydrogel microspheres, the empty rate of microspheres and loss rate of oocytes were determined. After releasing from microspheres and parthenogenetic activation with cytochalasin B and strontium chloride, the survival, cleavage and blastocyst rates were evaluated during in vitro maturation. Finally, oocytes encapsulated in sodium alginate microspheres were vitrified with low concentrations of cryoprotectants. We compared the survival and development capability of the oocytes with the Cryotop method. ResultsWhen the throat of the microfluidic chip measures 300 μm in length and 120 μm in width, microspheres can be uniformly formed at the throat of the chip. Sodium alginate generates microspheres with a wide size distribution when cross-linking outside the chip, while internal cross-linking within the chip results in more uniform microspheres. The stability of microsphere formation is significantly improved with the use of a three-channel internal cross-linking chip. At a flow rate of 2 μl/min and with 1% sodium alginate, the microfluidic chip can consistently and uniformly produce microspheres. Under flow rate ratios of 10, 15, and 20, the average microsphere diameters are 262.71 μm, 193.63 μm, and 156.63 μm, respectively. The sodium alginate hydrogel microspheres maintained their volume and structural integrity during the cooling and warming processes. Using a three-channel internal cross-linking microfluidic chip to encapsulate oocytes, at a flow rate ratio of 10, the empty rate is 32.28%, and the cell loss rate is 11.09%. After encapsulation and subsequent release, the oocyte survival rate (96.99%), cleavage rate (88.71%), and blastocyst formation rate (26.29%) showed no significant differences compared to the fresh group. After the microspheres were vitrified using a low concentration of cryoprotectant (10% DMSO+10% ehylene glycol (EG)+0.5 mol/L trehalose), the survival rate, cleavage rate, and blastocyst rate were 92.48%, 70.80%, and 20.42%, respectively. No significant difference was observed when compared to the Cryotop method using a higher concentration of cryoprotectant solution (15% DMSO+15% EG+0.5 mol/L trehalose). ConclusionWe designed and fabricated a microfluidic system with three-channel internal cross-linking chips used for oocyte vitrification preservation. The microfluidic system can generate oocytes-loaded sodium alginate hydrogel microspheres with uniform size, low empty rate, and good cold-resistance. The method successfully reduced the concentration of cryoprotectants in a single-step vitrification process, the developmental capability of oocytes during in vitro maturation were comparable with Cryotop method. Unlike the Cryotop method, the oocytes encapsulated in hydrogel does not come into contact with liquid nitrogen, eliminating the risk of cross-contamination. This study provides a novel approach to oocyte vitrification.

Estudio exploratorio de las motivaciones para la criopreservación de ovocitos en forma electiva / An exploratory study of motivations for elective oocyte cryopreservation

INTRODUCCIÓN: La vitrificación de óvulos es un avance científico que ha cambiado la perspectiva reproductiva de la sociedad humana. Este procedimiento se ha ofrecido como alternativa a la postergación voluntaria del embarazo, confiriéndole a la mujer una nueva perspectiva en su autonomía reproductiva. El número de mujeres que consultan y luego optan por congelar ovocitos ha aumentado en forma casi exponencial en Chile y en todo el mundo. En nuestro país, hay poco conocimiento acerca de la motivación, experiencia y resultados de la criopreservación electiva de ovocitos en Chile. El objetivo fue conocer la motivación, experiencia y el deseo reproductivo futuro de este grupo de mujeres sometidas a esta técnica. MÉTODOS: Estudio descriptivo transversal, basado en un cuestionario enviado por correo electrónico en el que participaron mujeres que se habían sometido previamente a criopreservación electiva de ovocitos entre enero de 2011 y diciembre de 2019 en Clínica Alemana, Santiago de Chile. RESULTADOS: De 342 mujeres que habían completado un ciclo de criopreservación electiva de ovocitos, 193 aceptaron participar y de estas, 98 (51%) de las mujeres contestaron la encuesta en forma satisfactoria. Se establecieron criterios de exclusión a aquellas mujeres que se habían sometido a este procedimiento por indicación médica como la endometriosis, el cáncer y la baja reserva ovárica. El motivo más frecuente para realizarse el procedimiento fue la edad (44%). En relación al procedimiento; el 94% no se arrepiente de haberlo realizado y 74% de las mujeres cree que utilizará sus ovocitos en algún momento de su vida. Por último, desde que se realizaron la criopreservación de ovocitos a la fecha, el 11% de las mujeres encuestadas ha usado sus ovocitos vitrificados y 27% ha logrado embarazarse con estos. CONCLUSIÓN: Las mujeres que se someten a criopreservación electiva de ovocitos por razones sociales, son principalmente mujeres sin pareja que tiene como motivación principal su edad reproductiva y la gran mayoría de ellas no se arrepienten de haberlo realizado.

INTRODUCTON: Oocyte vitrification is a scientific advance that has changed the reproductive perspective of human society. This procedure has been offered as an alternative to the voluntary postponement of pregnancy, giving women a new perspective on their reproductive autonomy. The number of women who consult and then choose to freeze oocytes has increased almost exponentially in Chile and throughout the world. There is little knowledge about the motivation, experience, and results of elective oocyte cryopreservation in Chile. The objective was to know the motivation, experience, and future reproductive desire of the women who underwent this technique. METHODS: Cross-sectional descriptive study based on a questionnaire sent by e-mail in which females who had previously undergone elective oocyte cryopreservation between January 2011 and December 2019 at Clínica Alemana, Santiago, Chile, participated. RESULTS: Of 342 women who had completed a cycle of elective oocyte cryopreservation, 193 agreed to participate, and of these, 98 (51%) answered the survey satisfactorily. Women who underwent this procedure for medical indication, including endometriosis, cancer, and low ovarian reserve, were excluded. The most frequent reason for the procedure was age (44%). Concerning the procedure: 94% do not regret having it done, and 74% of the women believe that they will use their oocytes at some point in their lives. Finally, from the time of oocyte cryopreservation to date, 11% of the surveyed women have used their vitrified oocytes, and 27% have become pregnant. CONCLUSIONS: Women who undergo elective oocyte cryopreservation for social reasons are mainly women without a partner whose main motivation is their reproductive age. The vast majority do not regret doing so.

Droplet freeze-thawing system based on solid surface vitrification and laser rewarming / 生物医学工程学杂志

Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 3 °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 6 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos

Abstract This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Resumo Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P 0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P 0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P 0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Ex vivo retrieval of mature oocytes for fertility preservation in a patient with bilateral borderline ovarian tumor / Recuperação ex vivo de oócitos maduros para preservação da fertilidade em paciente com tumor ovariano borderline bilateral

Abstract We report a case of ultrasound-guided ex vivo oocyte retrieval for fertility preservation in a woman with bilateral borderline ovarian tumor, for whom conventional transvaginal oocyte retrieval was deemed unsafe because of the increased risk of malignant cell spillage. Ovarian stimulation with gonadotropins was performed. Surgery was scheduled according to the ovarian response to exogenous gonadotropic stimulation; oophorectomized specimens were obtained by laparoscopy, and oocyte retrieval was performed ~ 37 hours after the ovulatory trigger. The sum of 20 ovarian follicles were aspirated, and 16 oocytes were obtained.We performed vitrification of 12 metaphase II oocytes and 3 oocytes matured in vitro. Our result emphasizes the viability of ex vivo mature oocyte retrieval after controlled ovarian stimulation for those with high risk of malignant dissemination by conventional approach.

Resumo Relatamos um caso de obtenção ex vivo de óvulos, guiada por ultrassonografia, para preservação da fertilidade em uma mulher com tumor ovariano borderline bilateral, para quem a recuperação transvaginal convencional foi considerada insegura, devido ao aumento do risco de disseminação de célulasmalignas. Foi realizada estimulação ovariana com gonadotrofinas. A cirurgia foi agendada de acordo com a resposta ovariana à estimulação gonadotrófica exógena; após ooforectomia por laparoscopia, ~ 37 horas após a maturação folicular, procedeu-se à recuperação extracorpórea de oócitos. Umtotal de 20 folículos ovarianos foi aspirado e 16 complexos cumulus foramobtidos, resultando na vitrificação de 12 oócitos maduros e de 3 oócitos imaturos amadurecidos in vitro. Nosso resultado enfatiza a viabilidade da recuperação ex vivo de oócitos maduros após estimulação ovariana controlada para mulheres com alto risco de disseminação maligna pela captação oocitária realizada convencionalmente pela via transvaginal.

Improving native human sperm freezing protection by using a modified vitrification method / 亚洲男科学杂志(英文版)

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing.

Research progress of methods to promote the survival of cryopreserved ovarian tissue auto-transplantation / 器官移植

With the technology development of cancer treatment, the survival rate of patients with cancer has been significantly improved. However, chemotherapy and radiation therapy may lead to premature ovarian failure and infertility in young women with cancer. Cryopreserved ovarian tissue auto-transplantation is an effective method to preserve fertility of such female patients. At present, the biggest challenge of this technique is mass loss of follicles after transplantation. In this article, the influencing factors and improvement methods of survival of cryopreserved ovarian tissue auto-transplantation were reviewed.

Effects of Magnetic Rewarming on Mechanical Properties of Vitrified Umbilical Artery / 医用生物力学

Objective To study the effect of magnetic rewarming on the morphology and biomechanical properties of vitrified umbilical artery. Methods The vitrified umbilical artery was rewarmed by magnetothermal method and traditional water bath. The temperature distribution and stress in the solution system were analyzed, and the rewarming effect was evaluated by tissue staining and mechanical test. Results Compared with water bath rewarming, the temperature gradient and thermal stress generated by magnetic rewarming were smaller, which could effectively reduce the thermal stress damage during the rewarming stage and achieve rapid and uniform rewarming. Magnetic rewarming could effectively avoid umbilical artery fractures and micro-cracks. After rewarming, the extracellular matrix, collagen fibers, elastic fibers and muscle fibers of the umbilical artery were evenly distributed, which preserved the macro and micro structures of the umbilical artery. The umbilical artery showed different degrees of hardening after water bath and magnetic rewarming, but the elastic modulus and limit stress of the latter were not significantly different from those of fresh umbilical artery, and the latter had unidirectional stretching characteristics similar to that of fresh umbilical artery, showing good elasticity and toughness. Conclusions Compared with water bath rewarming, magnetothermal method can effectively reduce the damage of rewarming stage, ensure the macroscopic, microscopic structure integrity of umbilical artery and better biomechanical properties. The research findings provide important references for cryopreservation of large tissues or organs such as umbilical artery.

Effects of vitrification and cryostorage duration on single-cell RNA-Seq profiling of vitrified-thawed human metaphase II oocytes / 医学前沿

Oocyte cryopreservation is widely used for clinical and social reasons. Previous studies have demonstrated that conventional slow-freezing cryopreservation procedures, but not storage time, can alter the gene expression profiles of frozen oocytes. Whether vitrification procedures and the related frozen storage durations have any effects on the transcriptomes of human metaphase II oocytes remain unknown. Four women (30-32 years old) who had undergone IVF treatment were recruited for this study. RNA-Seq profiles of 3 fresh oocytes and 13 surviving vitrified-thawed oocytes (3, 3, 4, and 3 oocytes were cryostored for 1,2, 3, and 12 months) were analyzed at a single-cell resolution. A total of 1987 genes were differentially expressed in the 13 vitrified-thawed oocytes. However, no differentially expressed genes were found between any two groups among the 1-, 2-, 3-, and 12-month storage groups. Further analysis revealed that the aberrant genes in the vitrified oocytes were closely related to oogenesis and development. Our findings indicated that the effects of vitrification on the transcriptomes of mature human oocytes are induced by the procedure itself, suggesting that long-term cryostorage of human oocytes is safe.

Effects of Vitrification with Self-made Carriers and Slow Programmed Freezing on Ovarian Tissue of Sheep / 中国医学科学院学报

Objective To investigate the effects of self-made carriers on the cryopreservation of ovarian tissue of sheep. Methods Thirty-two ovaries were randomly assigned to fresh group,programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group.The morphology,proliferation,apoptosis,and estrogen level of the ovarian tissue in each group were observed. Results After cryopreservation,the morphology normal rate of the primordial follicles in programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group were 74.2%,72.8%,and 72.3%,respectively,lower than that(83.7%)in the fresh group(χ

Freeze-all policy for in vitro fertilization in women with normal response to ovarian stimulation / Congelamento de todos os embriões em ciclos de fertilização in vitro em mulheres com resposta normal à estimulação ovariana

ABSTRACT Objective To answer the question if the freeze-all strategy and subsequent frozen embryo transfer is preferable to fresh embryo transfer for patients with normal response to ovarian stimulation (4 to 15 oocytes recovered) during in vitro fertilization treatments. Methods A retrospective cohort from two human reproduction centers between 2013 and 2017. A total of 471 frozen embryo transfers from freeze-all cycles, and 3,208 fresh transfers were included. Results After propensity score matching adjustment for age and number of eggs, 467 freeze-all cycles and 934 fresh cycles were analyzed, revealing no statistically significant difference between groups in relation to clinical pregnancy rate (32.5% in the Freeze-all Group and 32.3% in the Fresh Group, p=0.936). For women aged 40 years and older, we observed a statistically significant higher clinical pregnancy rate when freeze-all strategy was used (29.3% in the Freeze-all Group and 19.8% in the Fresh Group, p=0.04). Conclusion Freeze-all strategy was not superior to fresh transfer for all patients with normal response to ovarian stimulation. However, women aged 40 years and older could benefit from this strategy. This deserves further investigation in future research, preferable in a prospective randomized study.

RESUMO Objetivo Responder à pergunta se a estratégia freeze-all (congelamento de todos os embriões) e subsequente transferência de embriões congelados é preferível à transferência de embriões a fresco em pacientes com resposta normal à estimulação ovariana (4 a 15 ovócitos coletados) durante tratamentos de fertilização in vitro . Métodos Coorte retrospectiva de dois centros de reprodução humana entre 2013 e 2017. No total, foram incluídas 471 transferências de ciclos com congelamento de todos os embriões, e 3.208 transferências a fresco. Resultados Após o ajuste do escore de propensão para idade e número de óvulos, foram analisados 467 ciclos com congelamento de todos os embriões e 934 ciclos a fresco, não havendo diferença estatisticamente significativa entre os grupos em relação à taxa de gravidez clínica (32,5% no Grupo Freeze-all e 32,3% no Grupo a Fresco, p=0,936). Para mulheres com 40 anos ou mais, observamos uma taxa de gravidez clínica estatisticamente maior quando foi utilizada a estratégia freeze-all (29,3% no Grupo Freeze-all e 19,8% no Grupo a Fresco, p=0,04). Conclusão A estratégia freeze-all não foi superior à transferência a fresco para todas as pacientes com resposta normal à estimulação ovariana. No entanto, mulheres com 40 anos ou mais podem ter algum benefício com essa estratégia. Isso justifica uma investigação mais aprofundada em pesquisas futuras e, de preferência, em estudos prospectivos randomizados.

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Vitrification-based cryopreservation of tissues: Strengths and existing problems / 中国组织工程研究

BACKGROUND: Vitrification-based cryopreservation is a promising cryopreservation method, which can change the state of the biological materials by using high concentration vitrification reagent to realize active preservation. OBJECTIVE: To review the biological principle of vitrification-based cryopreservation, the classification of cryopreservation reagents, as well as the cryopreservation of ovary skin, cornea and other medical tissue specimens. METHODS: PubMed and WanFang databases were searched for relevant articles published from January 1994 through October 2019. Search terms were “tissue; vitrification; cryopreservation” in English and Chinese, respectively. According to the inclusion and exclusion criteria, 45 articles were finally included in result analysis.RESULTS AND CONCLUSION: The process of vitrification-based cryopreservation can avoid ice crystallization by which the cells are damaged, and effectively preserve the cell’s biological activity and basic functions. Vitrified cryopreservation reagents can be divided into permeable and non-permeable reagents. Their operation is simple and efficient. The only disadvantage is that the high concentration of cryopreservation reagents can cause some toxic injuries to the cells. To reduce the risk of overall tissue damage, a variety of low-toxic cryopreservation reagents can be mixed and used. At present, vitrification-based cryopreservation technology has been successfully applied in a variety of cells. However, the technical problems in the cryopreservation of tissues have not been solved completely.

Vitrificación de ovocitos en pacientes oncológicas: experiencia en clínica ivi chile y revisión de la literatura / Vitrification of oocytes in oncological patients: experience in clinica ivi chile and review of literature

La sobrevida de pacientes con cáncer ha mejorado con el tiempo, especialmente en pacientes en edad fértil. La criopreservación de los ovocitos a través de la estimulación ovárica controlada (EOC) es la técnica más frecuente de preservación de la fertilidad. El objetivo del presente estudio es realizar un análisis descriptivo de los ciclos de pacientes que, previo al tratamiento de cáncer, realizaron un tratamiento de preservación de fertilidad. Se analizaron datos demográficos como edad, diagnóstico de ingreso y resultados clínicos, tales como tipo de protocolo de estimulación utilizado, número de ovocitos obtenidos, duración de la estimulación y momento de inicio en el ciclo. Resultados: La edad promedio fue 28.9 años. La duración media de la estimulación fue de 12 días, con un promedio de ovocitos obtenidos en total de 12. Se utilizaron 2 protocolos de estimulación ovárica, obteniendo mejores resultados con el esquema de antagonistas de GnRH asociado a letrozole y doble gatillante. Respecto al momento del ciclo en que se inició la estimulación ovárica, no hubo diferencias. Conclusiones: Es posible realizar preservación de la fertilidad previo a un tratamiento oncológico con buenos resultados en pacientes jóvenes, por lo que sugerimos realizarlo en todos los pacientes con diagnóstico oncológico antes el tratamiento del cáncer. Es recomendable comenzar la estimulación ovárica en cualquier fase del ciclo ya que se obtienen los mismos resultados y permite un pronto inicio de la terapia oncológica.

Survival of patients with cancer has been improving over time, especially in young patient with fertility intention. Cryopreservation of oocytes through controlled ovarian stimulation (EOC) is the most frequent technique of fertility preservation. We analyzed the data obtained from oncological patients who attended IVI Chile between January 2008 and May 2017 in search of fertility preservation. Demographic data were obtained: age, diagnosis of admission, type of stimulation protocol used, number of oocytes obtained, duration of stimulation and pregnancy rate. Results: The average age: 28,9 years; average duration of stimulation:12 days. Number of oocytes obtained in total: 12. Two ovarian stimulation protocols were used. The one with the best results was the protocol with GnRH antagonists associated with letrozole and double triggering. Regarding the moment of the cycle where to start ovarian stimulation, there were no differences. Conclusions: It is possible to carry out a fertility preservation treatment prior to an oncological treatment with good results in young patients, so we suggest the preservation of fertility in all patients with an oncological diagnosis before oncological treatment. It is recommended to start ovarian stimulation at any phase of the cycle since the same results are obtained.

Fresh versus frozen embryo transfer: a retrospective cohort study

Background: Elective frozen embryo transfer (FET), has recently increased significantly with improvements in cryopreservation techniques. Observational studies and randomized controlled trials suggested that the endometrium in stimulated cycles is not optimally prepared for implantation; risk of ovarian hyperstimulation syndrome reduced and pregnancy rates increased following FET and perinatal outcomes are less affected after FET. However, the evidence is not unequivocal and recent randomised control trials challenge the use of elective FET for the general IVF population. Pregnancy rates were analysed in a cohort of patients undergoing embryo transfers.Methods: This was a retrospective cohort study of patients who underwent embryo transfers from April 2018 to March 2019 at study centre in Surat.175 cycles of embryo transfers (119 fresh and 56 frozen) were included in the study. Outcomes measured were positive pregnancy, clinical pregnancy and ongoing clinical pregnancy rates achieved in the IVF-ET cycles.Results: There were no statistically significant differences between positive pregnancy rate (54.6% versus 60.7%, Odds ratio (OR) 0.78; 95% Confidence Interval (CI) 0.41-1.49), clinical pregnancy rate (48.73% versus 57.14%, OR 0.52; 95% CI 0.1- 2.64) and ongoing clinical pregnancy rate  (45.38% versus 51.78% OR 1.4; 95% CI 0.29 - 6.67) in fresh ET and FET cycles, respectively, p < 0.05 was considered statistically significant for all measures.Conclusions: Despite the observed higher rates of positive biochemical, clinical and ongoing clinical pregnancy per transfer in the FET cohort, these did not reach statistical significance. Thus, both transfer strategies are reasonable options, although there is a trend favouring the freeze-all strategy.

Case reports for embryo banking: for women who want their own

Out of the many challenges in management of female factor infertility, poor responders and low response to stimulation in aged and even younger women, seems to be a common problem. It is very difficult to offer one particular management strategy or treatment protocol for optimum outcome in this group of women of poor responders. In a low resource set up, IVF (In vitro Fertilization) specialist doctors usually face a challenge in treating women with poor/ low ovarian reserve as ovum / gamete donation is considered as a taboo in various sections of society even today. Hence women insist on having an offspring of "their own" and vehemently deny ovum / gamete donations. In this article we discuss 2 cases of poor ovarian reserve retrospectively, who underwent multiple cycles of controlled ovarian hyperstimulation for embryo banking and ultimately achieved pregnancy. Both patients achieved pregnancy with the method of embryo banking. Embryo banking should be considered and discussed. Various articles have discussed the advantages and disadvantages of embryo banking or even oocytes accumulation. The advantages of this technique is patients with poor/low ovarian reserve get a chance to be pregnant with their own oocytes and also have a chance for vitrification of residual embryos. Another advantage in such patients is that the embryos can undergo PGS (Preimplantation Genetic Screening) techniques in cases of suspected genetic disorders. The disadvantage in a low resource set up like India is the cost of the treatment. Nevertheless, embryo banking and accumulation of oocytes should be given as an option for treatment of poor/ low ovarian reserve and could be considered as a ray of hope for all future mothers hoping for a child of "their own".

Survival of isolated human preantral follicles after vitrification: Analyses of morphology and Fas ligand and caspase-3 mRNA expression / 대한생식의학회지

OBJECTIVE: This study aimed to examine the effect of vitrification on apoptosis and survival in human preantral follicles after thawing.METHODS: This experimental study was conducted at an acute tertiary care hospital from March 2012 to April 2013. Ovaries were sliced into 5×5×1-mm pieces and divided into the following three groups: preantral follicle isolation, ovarian tissue vitrification-warming followed by follicle isolation, and immunohistochemistry of fresh ovarian tissue. For statistical analyses, the Student t-test, chi-square test, Kruskal-Wallis test, and Kaplan-Meier survival analysis were used.RESULTS: A total of 161 preantral follicles (70% secondary) were collected from ovarian cortex tissue of six women between 30 and 37 years of age who underwent oophorectomy due to cervical cancer or breast cancer. There were no significant differences in the follicular morphology of fresh preantral follicles and vitrified follicles after thawing. The mean Fas ligand (FasL) mRNA expression level was 0.43±0.20 (relative to β-actin) in fresh preantral follicles versus 0.51±0.20 in vitrified follicles (p=0.22). The mean caspase-3 mRNA expression level in fresh preantral follicles was 0.56±0.49 vs. 0.27±0.21 in vitrified follicles (p=0.233). One vitrified-thawed secondary follicle grew and developed to an antral follicle within 6 days of culture.CONCLUSION: Vitrification did not affect preantral follicle morphology or mRNA expression of the apoptosis markers FasL and caspase-3. Further studies are required to establish whether vitrification affects the outcomes of in vitro culture and the maturation of preantral follicles.